Optical absorbance across a capillary flowcell is used to determine the presence and/or concentration of an analyte within the capillary.
The basic optical configuration of a multiple wavelength detector is different from that of a variable wavelength detector. However, each type of detector is often configured as a dual beam design, with a flowcell containing analyte(s) in one beam, and a reference flowcell in an alternate reference beam. Such designs permit the determination of lower concentrations of analyte by compensating, for example, for fluctuations in source energy, and reduction of signal variation as a function of wavelength. Electromagnetic radiation which is delivered through the analyte 16 and to the detector 22 (i.e., the signal) competes with electromagnetic radiation which is delivered to the detector 22 without having passed through the analyte 16 (i.e., "noise").
In a variable wavelength detector, broad-band optical radiation from a source is passed through a wavelength selection device such as a monochromator. A narrow, nearly monochromatic band of electromagnetic radiation is selected and focused upon the entrance aperture of the flowcell. Optical radiation which is not scattered or absorbed by the analyte(s) in the flowcell is transmitted through the flowcell, and is converted to an electrical signal by a detector element. If the detector element is sufficiently sensitive and of sufficient area to intercept the transmitted optical beam, it is not necessary to refocus the beam. Variable wavelength detectors of the prior art (which determine a single wavelength of electromagnetic radiation, s.lambda.), are shown in FIG. 1 and in FIG. 2.
FIG. 1 shows a diagrammatic representation of the path of light through a capillary flowcell in a variable wavelength detector. Monochromatic light at a single wavelength (s.lambda.) is emitted from an electromagnetic radiation source 10. Electromagnetic radiation (shown as arrows) enters the flowcell 12 by passing through a mask 13 at the entrance aperture 14. The mask 13 is placed to restrict optical radiation which would not pass through the inner diameter of the capillary (and thus the analyte) within the flowcell. The electromagnetic radiation passes through the capillary tube wall 16 and through the analyte 18 contained within the capillary tube. Electromagnetic radiation which has passed through the analyte 18 and which then passes through the exit aperture 20 is converted to an electrical signal by a detector element 22. The optical axis .alpha. of the flowcell system is shown for reference purposes.
FIG. 2 shows a diagrammatic representation of the path of light through an alternate capillary flowcell in a variable wavelength detector. Monochromatic light at a single wavelength (s.lambda.) is emitted from an electromagnetic radiation source 10. Electromagnetic radiation (shown as arrows) enters the flowcell 12 at the entrance aperture 14. A spherical ball lens 15 is then used to reimage the entrance aperture 14 onto the inner diameter of the capillary tube 16 containing the analyte 18. The focused electromagnetic radiation passes through the capillary tube 16 wall and through the analyte 18 contained within the capillary tube. Electromagnetic radiation which has passed through the analyte 18 and which passes through the exit aperture 20 is converted to an electrical signal by a detector element 22. The optical axis .alpha. of the flowcell system is shown for reference purposes.
In a multiple wavelength detector, a beam of optical radiation including a multiplicity of wavelengths is focused onto a flowcell. The analyte(s) in the flowcell may absorb optical radiation at a multiplicity of different wavelengths. Optical radiation which is not absorbed by the analyte(s) in the flowcell is transmitted through the flowcell, and through a polychromator. The polychromator disperses the transmitted optical radiation into many narrow bands, as a function of band wavelength. Each narrow band is focused onto a different detector element in a detector array, and is converted to an electrical signal. The absorbance of the analyte(s) in the flowcell is thus measured as a function of wavelength absorption or transmission of many different wavelengths. A multiple wavelength detector such as that found in the prior art, having a beam of multiple wavelength of electromagnetic radiation (m.lambda.), is shown in FIG. 3. Representative wavelengths (.lambda..sub.1, .lambda..sub.2, and .lambda..sub.3) are shown after separation by the polychromator.
FIG. 3 shows a diagrammatic representation of the path of light through a capillary flowcell in a multiple wavelength detector. Polychromatic light at various wavelengths (m.lambda.) is emitted from an electromagnetic radiation source 10. Electromagnetic radiation (shown as arrows) enters the flowcell 12 by passing through a mask 13 at the entrance aperture 14. The mask 13 is placed to restrict optical radiation which would not pass through the inner diameter of the capillary (and thus the analyte 18) within the flowcell. The electromagnetic radiation passes through the capillary tube wall 16 and through the analyte 18 contained within the capillary tube. Electromagnetic radiation which has passed through the analyte 18 and which passes through the exit aperture 20 is split into discrete bands (such as .lambda..sub.1, .lambda..sub.2 and .lambda..sub.3) by the polychromator 24. As shown, the polychromator can include an entrance aperture 21. Alternatively, the exit aperture 20 of the flowcell 10 can act as the entrance aperture for the polychromator. Each discrete wavelength of electromagnetic radiation is then converted to an electrical signal by discrete detector elements 22. The optical axis .alpha. of the flowcell system is shown for reference purposes.
The fundamental design differences between variable wavelength detectors and multiple wavelength detectors place conflicting demands upon flowcell design, especially with respect to optical radiation transmitted through the analyte. Variable wavelength detectors are often designed such that the beam of optical radiation does not require focusing after passing through the analyte. Rather, the beam is uninterrupted between the capillary containing the analyte and the detector element. Multiple wavelength detectors generally require that the beam be refocused for the polychromator after the beam has passed through the analyte sample. The exit aperture of the flowcell can serve as the entrance aperture for the polychromator.
The presence of a capillary column within the flowcell restricts flowcell design. Because chromatic integrity is maintained by minimizing off-column chromatic dispersion, on-column detection is usual. In on-column detection, the capillary forms part of the flowcell.
Albin et al., "Fluorescence Detection in Capillary Electrophoresis: Evaluation of Derivatizing Reagents and Techniques", Anal. Chem. 63 (1991) describe a capillary electrophoresis fluorescence detector. A single sapphire lens is used to focus the monochromatic beam before the beam is passed through the capillary flowcell.
Kobayashi et al. ("Photodiode Array Detection in High-Performance Capillary Electrophoresis", J. Chrom. 480 (1989):179-184) describe replacement of a standard cell of a spectrometer with a capillary. The light intensity of the apparatus was decreased to prevent saturation of the diode array.
Moring et al., "Analytical Aspects of an Automated Capillary Electrophoresis System", LC:GC 8(1):34-46 (1990) describes a variable wavelength detector in which one sapphire lens is used to focus the monochromatic light prior to the time the beam is focused on the capillary flowcell.
A capillary flowcell is described in Sepaniak et al., "Instrumental Developments in Micellar Electrokinetic Capillary Chromatography", J. Chrom. 480 (1989):185-196. The capillary flowcell is produced by removing a portion of the capillary coating, epoxying the capillary between two microscope slides, attaching the capillary and slides to a bracket, painting the front face of the apparatus black, and using an argon laser to etch the painted section. The flowcell is completed by masking all but a small portion with electrical tape. The flowcell is described for use with each of a variable wavelength detector and a multiple wavelength detector. When the flowcell is used in a multiple wavelength detector, a beam of optical radiation originates in a deuterium lamp, passes first through the capillary, and then through two separate lenses before it is focused at the entrance of the spectrometer.